We will investigate the replication mechanisms of the maxicircle component of the kinetoplast DNA (kDNA) of the trypanosome Crithidia fasciculata. Autonomous replication sequences (ARS) present in maxicircle DNA will be selected by cloning into yeast plasmids carrying a selectable yeast gene but lacking the capability for autonomous replication. Cloned ARSs will be sub-cloned to identify the minimal functional sequence and to allow rapid DNA sequence analysis. Plasmids carrying a selectable drug-resistance gene will be constructed to allow the development of DNA transformation techniques for trypanosomes and to provide appropriate vectors for cloning genes involved in the replication of kDNA. Cloned ARSs will also be used to identify proteins that interact specifically with maxicircle ARSs and as templates for studies of the in vitro replication of maxicircle DNA.